This invention is generally in the field of drug delivery and more specifically in the area of fibrin and synthetic matrices to enhance wound healing.
Fibrin matrices are present naturally in the body and serve as the initial matrix for wound healing. When an injury occurs to tissue, blood vessels are compromised, allowing the precursor molecule, fibrinogen, to invade the wound. The fibrinogen is then enzymatically cleaved and self-catalyzed into a loosely formed gel. The gel is then covalently crosslinked through the action of the transglutaminase, factor XIIIa, resulting in a stable matrix. Pisano, Finlayson and Peyton, Science, 160, 892-893 (1968).
In vivo, the final fibrin matrix includes various proteins in addition to fibrinogen, such as serum proteins present during the coagulation process, for example fibronectin and α2-plasmin inhibitor. Factor XIIIa can covalently crosslink these serum proteins to the fibrin matrix, which can then add additional bioactivities to the matrix that can modify the ability of cells to infiltrate and degrade the matrix. Tamaki and Aoki, J Biol Chem, 257, 14767-14772 (1982). These matrices also contain many blood cells, which become entrapped inside the matrix during coagulation, further modifying the biochemical character of the matrix. One major cell type is the platelet, a cell rich with natural supplies of potentially therapeutic growth factors.
One key advantage of fibrin is that it is a matrix that is strongly conductive for cells, allowing them to easily infiltrate the wound site. The process employed involves two key features. First, the matrix contains adhesion sites, allowing the cells to attach and migrate into the gel. Additionally, the matrix is responsive to cell-derived proteolytic activity. This allows the matrix to be degraded locally, allowing the cells to migrate into the matrix uninhibited but preventing global degradation of the matrix. Herbert, Bittner and Hubbell, J Compar Neuro, 365, 380-391 (1996); Pittman and Buettner, Dev Neuro, 11, 361-375 (1989). Therefore, the natural matrix remains at the site of injury until it is infiltrated by cells, at which time it is degraded during this process leading to regenerated tissue.
The natural healing process is sometimes inadequate, such as when this general healing response fails to lead to regeneration of functional specialized tissue. See for example, Robello G T and Aron D N, Semin Vet Med Surg (Small Anim), 7, 98-104 (1992). Therefore, there is a need for a means to induce formation of complete, functional regenerated tissue, especially regenerated specialized tissue.
Many bioactive molecules, including growth factors, peptides, and other assorted molecules, have been discovered which can affect tissue regeneration. Schense and Hubbell, Bioconj Chem, 10, 75-81 (1999). Previous work has shown that growth factors can be precipitated within a fibrin matrix. MacPhee, Druhan et al., 76 (1995); U.S. Pat. Nos. 6,117,425 and 6,197,325 to MacFee, et al. However, these investigators have not recognized the strong advantages of working with non-glycosylated growth factors, and especially non-glycosylated members of the cystine knot growth factor superfamily, in particular of the TGFβ superfamily.
Growth factors play an important role in wound healing, and are often naturally present at the site of injury. However if growth factors are applied to the body in high concentrations, adverse effects are likely to be observed. For example, if the retention mechanism of bone morphogenetic protein (BMP) in a matrix is not optimized, i.e. if the BMP simply diffuses from the matrix within the first hours, high doses of BMP in the matrix are necessary to cause a local response at the site of injury. As a result most of the BMP circulates freely in the body and ectopic bone formation may occur. It is therefore necessary to keep the freely circulating concentration of the growth factor as low as possible, while maintaining a concentration which is sufficiently high locally so that the desired therapeutic response is triggered at the site of injury. Some growth factor receptors must be occupied for at least 12 hours to produce a maximal biologically effect. Therefore, a prolonged contact caused by a small but constant stream of growth factor near the site of need is very favorable for a healing response. At the same constant release rate, as the initial concentration of growth factor retained in the matrix increases, the time period for release from the matrix increases.
Therefore it is an object of the present invention to increase the retainable concentration of bioactive molecules, in particular growth factors, in a matrix.
A further object of the present invention is to provide a method to decrease the solubility of a growth factor in a matrix made from fibrin or synthetic polymers.
It is still a further object of the present invention to provide compositions and methods for making compositions to improve wound healing.